It is well known that oscillations of intracellular Ca2+ control the activity of the ubiquitous and essential signaling protein, calmodulin (CaM). It is les widely recognized that CaM is also regulated by two small proteins, PEP-19 and neurogranin (Ng), which have no known intrinsic activity other than binding to CaM via an IQ motif. The mechanisms of action of these Regulators of CaM Signaling, or RCS proteins, are poorly understood even though they broadly affect CaM signaling to provide a level of control that is analogous to RGS and GAPs in G-protein signaling. The biological roles of RCS proteins are profound and diverse. Ng knockout mice exhibit learning deficits, deficiencies in synaptic plasticity and altered Ca2+ levels and dynamics. In addition, Ng levels are down regulated in hypothyroidism, Alzheimer disease and schizophrenic patients. PEP-19 is expressed in the central nervous system, but also in renal/urinary and reproductive tissues, and its levels are significantly altered during normal cell processes such as B-cell anergy, and by pathological conditions such as cancer and Huntington's disease. In addition, increased expression of PEP-19 inhibits apoptosis, protects against cell death due to Ca2+ overload, and causes premature neuronal differentiation in transgenic mice. Despite these profound biological impacts, the prevailing mechanistic model that RCS proteins simply buffer CaM does not explain their different cellular effects, or account for their important and divergent biochemical properties. This proposal provides new hypotheses based on our recent and preliminary data showing that Ca2+ exchange at the C-domain of CaM is greatly accelerated by PEP-19, while the Ca2+ binding affinity is greatly reduced by Ng, and that this is mediated by acidic sequences adjacent to the IQ motifs. We also show that PEP-19 sensitizes HeLa cells to ATP-dependent Ca2+ release, and that this dependent on its ability to modulate the Ca2+ binding properties of CaM. These data form the foundation for the following Aims: Aim 1: Define the structural basis for differential effects of RCS proteins on Ca2+ binding to CaM. Aim 2: Determine if RCS proteins play a general role in regulating ligand-dependent Ca2+ release. Aim 3: Identify properties of RCS proteins that mediate their effects on apoptosis